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Extracellular matrix stiffness and composition jointly regulate the induction of malignant phenotypes in mammary epithelium

Nature Materials volume 13, pages 970–978 (2014)Cite this article

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Abstract

In vitro models of normal mammary epithelium have correlated increased extracellular matrix (ECM) stiffness with malignant phenotypes. However, the role of increased stiffness in this transformation remains unclear because of difficulties in controlling ECM stiffness, composition and architecture independently. Here we demonstrate that interpenetrating networks of reconstituted basement membrane matrix and alginate can be used to modulate ECM stiffness independently of composition and architecture. We find that, in normal mammary epithelial cells, increasing ECM stiffness alone induces malignant phenotypes but that the effect is completely abrogated when accompanied by an increase in basement-membrane ligands. We also find that the combination of stiffness and composition is sensed through β4 integrin, Rac1, and the PI3K pathway, and suggest a mechanism in which an increase in ECM stiffness, without an increase in basement membrane ligands, prevents normal α6β4 integrin clustering into hemidesmosomes.

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Figure 1: The stiffness of interpenetrating networks of alginate and basement-membrane matrix can be modulated independently of cell-adhesion-ligand density for 3D cell culture.
Figure 2: Enhanced stiffness alone leads to the malignant phenotype in MCF10As.
Figure 3: Altered ECM composition can enhance or completely abrogate the effect of increased stiffness on the phenotype of MCF10As.
Figure 4: Mechanotransduction and malignant phenotype of MCF10As in IPNs is mediated through β4 integrin signalling.
Figure 5: Mechanotransduction and malignant phenotype of MCF10As in stiff IPNs is mediated through the PI3K signalling pathway and Rac1 activation.
Figure 6: Proposed mechanism for the impact of ECM stiffness and composition on malignant phenotype.

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Acknowledgements

The authors acknowledge the help of A. Li, D. Klumpers, A. Mao and other members of the Mooney lab. The authors also thank J. Brugge (Harvard Medical School) for providing the β4-integrin and Rac1 mutant plasmids, L. Lichten (Qiagen) for help with RNA arrays, Louise Jawerth/Weitz lab for help/use of the rheometer, M. Ericsson and L. Trakimas of the Harvard Medical School EM facility for help with transmission electron microscopy, P. Mali (Harvard Medical School) for discussions, and the Bauer Core for flow sorting. This work was supported by an NIH F32 grant to O.C. (CA153802), fellowships from NSERC and HHMI for S.T.K., fellowships from FCT, FCG and FLAD for C.B.d.C., and NIH (R01EB015498) and MRSEC (DMR-0820484) grants to D.J.M. This work was performed in part at the Center for Nanoscale Systems (CNS), a member of the National Nanotechnology Infrastructure Network (NNIN).

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Authors and Affiliations

  1. School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138, USA

    Ovijit Chaudhuri, Sandeep T. Koshy, Cristiana Branco da Cunha, Jae-Won Shin, Catia S. Verbeke & David J. Mooney

  2. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, USA

    Ovijit Chaudhuri, Sandeep T. Koshy, Cristiana Branco da Cunha, Jae-Won Shin, Catia S. Verbeke & David J. Mooney

  3. Department of Mechanical Engineering, Stanford University, Stanford, California 94305, USA

    Ovijit Chaudhuri

  4. Harvard-MIT Division of Health Sciences and Technology, Cambridge, Massachusetts 02139, USA

    Sandeep T. Koshy

  5. Institute of Molecular Pathology and Immunology, Instituto de Engenharia Biomédica, and Faculty of Medicine of the University of Porto, Porto 4150-180, Portugal

    Cristiana Branco da Cunha

  6. Department of Pathology, Stanford University Medical Center, Stanford, California 94305, USA

    Kimberly H. Allison

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Contributions

O.C. and D.J.M. designed the IPNs. O.C., S.T.K. and D.J.M. designed the experiments. O.C., S.T.K., C.B.d.C., J-W.S. and C.S.V. conducted experiments and analysed data. C.B.d.C. designed and conducted the RNA expression arrays. K.H.A. conducted the comparison of in vitro results to human-breast-cancer samples. O.C., S.T.K., C.B.d.C. and D.J.M. wrote the manuscript.

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Correspondence to David J. Mooney.

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Chaudhuri, O., Koshy, S., Branco da Cunha, C. et al. Extracellular matrix stiffness and composition jointly regulate the induction of malignant phenotypes in mammary epithelium. Nature Mater 13, 970–978 (2014). https://doi.org/10.1038/nmat4009

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